Human Immunodeficiency Virus (HIV) is known as the causative agent of Acquired Immunodeficiency Syndrome (AIDS). Especially, HIV-1 and HIV-2 belongs to the subgroup Lentivirdae of retroviruses, which carry a single-stranded RNA. Once in a susceptible host cell, the RNA genome is reverse-translated by viral reverse transcriptase to generate double-stranded DNA, which becomes integrated into the host's chromosome, resulting in provirus.
In order to better understand the background of the present invention, with reference to FIG. 1, there is given the structure of the HIV-1 genome 9.2 kb in size. The HIV-1 genome possesses structural genes consisting of gag, pol and env, and accessory genes including tat, rev, nef, vif, vpu and vpx.
A HIV Gag polyprotein precursor, Pr55gag, is expressed from full-length gag-pol RNA with a size of 55 kDa. A HIV Gag precursor is transported to the inner surface of the cell plasma membrane by a N-terminal myristate moiety, where directing the assembly of virions. Maturation of virions requires the proteolytic cleavage of the Pr55gag by protease (Pro) encoded by the pol gene of HIV into matrix (MA, p17), capsid (CA, p24), nucleocapsid (NA, p9) and p6 proteins (Gheysen D et al, Cell, 59, 103-112 (1989); Bray, M. et al. Proc. Natl. Acad. Sci. USA 91, 1256-1260 (1994); Cann, A. J., and J. Karn, AIDS 3 (Suppl.1), S19-S34 (1989); Ratner, L., W. et al., Nature 313, 277-284 (1985); Wain-Hobson, S. et al., Cell 40, 9-17 (1985)).
The envelope glycoprotein (Env) precursor, gp160, is expressed from the spliced RNA. In virus-infected cells, the precursor is cleaved by protease from the host or the virus into gp120 subunit at the N-terminal side and gp41 subunit at the C-terminal side, which are properly targeted to a plasma membrane with anchorage on the external surface and insertion into the cell membrane, respectively. In the mature virion, gp120 and gp41 subunits form a heterodimer in which the two subunits are non-covalently associated, which is known to be directly responsible for the infectivity, cellular tropism and cytopathogenicity of HIV (Robey, E. and Axel, R., Cell 60, 697-700 (1990)). Because of the close relation to immunogenicity, the Env protein of HIV is a target for the development of the HIV vaccines.
The protease (Pro) is produced from self-cleavage of Pr160 gag-pol precursor, translated by ribosomal frame shift at 3′ end of gag mRNA region while translating gag-pol mRNA (Jacks, T. and Varmus, H. E. Science 230, 1237-1242 (1985); Jacks, T. et al, Nature 331, 280-283 (1988); Sonigo, p. et al, Cell 45, 375-385 (1986)). The protease participates in the processing of the Pr160gag-pol polyprotein and env protein, resulting in mature infectious virions (Kohl, N. et al., Proc. Natl. Acad. Sci. USA 85, 4686-4690 (1988)).
To data, suggested AIDS vaccines include inactivated vaccines, attenuated vaccines, recombinant live vaccines, virus-like particle vaccines, and subunit vaccines.
The inactivated whole virus vaccines are prepared by chemically or physically treating isolated viral particles to destroy their infectivity. This kind of vaccine is easy to prepare and is capable of using most epitopes, in contrast, it may cause diseases when the virus is not completely inactivated, on the other hand, the strong treatment for the complete inactivation may cause destruction of many epitopes. Although there are some successful reports on the inactivated SIV vaccines (Scott E. J. Nature 253, 393 (1991); Le Grand R. et al., Nature 355, 684 (1992); Crange M. P. et al., Nature 355, 685-686 (1992); Arthur L. O. et al., J Virol 69, 3117-3124 (1195); Neidrig M. et al., Vaccine 11, 67-74 (1993)), it is not considered useful as a safe and effective AIDS vaccine because suspicion has been raised that the successful immunization resulted from action by xeno-antigen which the cultivated host cells harbor.
The live attenuated vaccines are prepared by partial deletion in the HIV genome, such as deletion of nef gene or nef and vpr genes (Descrosiers R. C. AIDS Res Hum Retroviruses 8, 411-421 (1992); Gibbs J. S. et al., AIDS Res Hum Retroviruses 10, 607-616 (1994); Cranage M. P. et al., Virology 229, 143-154 (1997); Wyand M. S. et al. Nature Med 3, 32-36 (1997)). However, such vaccines can regain the deleted genes in vaccinated animals, allowing them to be infectious. Accordingly, the live attenuated vaccines may not be completely safe because they can cause AIDS in vaccinated individuals (or animals).
The live recombinant vaccines are vectors in which nonessential genes of non-pathogenic viruses are replaced with genes coding HIV immunogens. There was disclosed an expression of gag, pol or env gene and an expression of an immunogenic region including V3 loop of HIV as a chimeric form in vaccinia virus, poxvirus, canary pox virus, avipox virus and poliovirus, or influenza virus (Tartaglia J. et al., Virology, 188, 217-232 (1992); Abimiku A. et al., Nature Med, 1, 321-329 (1995); Natuk R. J. et al., AIDS Hum Retroviruses 9, 395-404 (1993); Li S. et al., J Virol, 67, 6659-6666 (1993); Muster T. et al., J Virol 69, 6678-6686 (1995)). As this technology, in general, uses a part of the protein as a vaccine material, it cannot provide an efficient immunogen. In addition, the live recombinant vectors are unstable (Morrow, C. D. et al., AIDS Res Hum Retroviruses, 10, S61-S66 (1994); Anderson M. J. et al., AIDS Res Hum Retroviruses, 13, 53-62 (1997)).
The subunit vaccines are subunits of proteins encoded by HIV, which are naturally isolated or expressed by recombinant DNA technology. These vaccines can be easily manufactured, but they have a different conformation from original proteins existing in viral particles. Recombinant Env glycoproteins, gp120 and gp160, were expressed using mammalian and insect cell expression systems (Lasky, L. A., et al., Science, 249, 932-935 (1986); Barr, P. J. et al., Vaccine 5, 90-101 (1987); Hu, S-L. et al., J Virol 61, 3617-3620 (1987); Rusche, J. R. et al., Proc. Natl. Acad. Sci., 84, 6924-6928 (1987); Berman, P. W., et al. J Virol 63, 3489-3498 (1989); Ivey-Hoyle, M., and Rosenberg, M., Mol. Cell. Biol., 10, 6152-6159 (1990); Lasky, L. A. et al., Cell, 50, 975-985 (1987)). Upon being injected into chimpanzees, gp120 subunit vaccine expressed in the CHO cells could not confer protection against a viral challenge experiment (Berman, P. W. et al., Proc. Natl. Acad. Sci., 85, 5200-5204 (1988)).
Generally, vaccines of viral particle form are more immunogenic and stable than those of subunit form. There was recently developed a new technology in which the non-infectious virus-like particles (VLPs) that do not carry genetic materials were used as vaccines. For example, mutation in packaging genes can provide a VLP without an RNA genome, and a VLP containing unprocessed gag protein can be prepared using recombinant baculovirus or vaccinia virus (Gorelink R. J. et al., J Virol 64, 3207-3211 (1990); Gheysen D. E. et al., Cell 59, 103-112 (1989)). It was also reported that virus-like particles were produced from the cells co-transfected with two recombinant vaccinia viruses containing gag-pol gene and env gene, respectively (Haffar O. K. et al., Virology; 183, 487-495 (1991)). Such pseudo-particles were effective for production of neutralizing antibodies, env-CD4 protecting antibodies, and syncytium inhibition, but they have the drawbacks in their not being mature virus particles and lack of infectivity.
As other forms of virus-like particles containing immunodeterminant of HIV, there were described hybrid particles including poliovirus capsovector, hepatitis B virus (HBV) core particles, HbsAg particles and yeast retrotransposon virus-like particles But, these hybrid particles contain only the partial region of the immunodeterminant of HIV, resulting in non-effective vaccines (Grene E. et al., AIDS Res Hum Retroviruses, 13, 41-51 (1997); Schlienger K., et al. J Virol 66, 2570-2576 (1991); Adams S. E. et al., Nature 329, 68-70 (1987); Layton G. T. et al., J Immunol, 151, 1097-1107 (1993); Eckart L. et al., J Gen Virol 77, 2001-2008 (1996)).
The inventors of the present invention attempted to develop a novel AIDS vaccine with mature virus-like particles using an alphavirus vector system. The inventors believe the virus-like particles will provide the way to prevent AIDS.